1. Field of the Invention
This invention is in the field of tissue engineering. The invention is directed to collagenous tissues which have been treated to remove non-collagenous components such as cells, cellular debris, and other extracellular matrix components, such as proteoglycans and glycosaminoglycans, normally found in native tissues. Treatment of the tissue with alkali, chelating agents, acids and salts removes non-collagenous components from the collagenous tissue matrix while controlling the amount of swelling and dissolution so that the resultant collagen matrix retains its structural organization, integrity and bioremodelable properties. The process circumvents the need to use detergents and enzymes which detrimentally affect the cell compatibility, strength and bioremodelability of the collagen matrix. The collagenous tissue matrix is used for implantation, repair, or use in a mammalian host.
2. Brief Description of the Background of the Invention
The field of tissue engineering combines the methods of the engineering with the principles of life sciences to understand the structural and functional relationships in normal and pathological mammalian tissues. The goal of tissue engineering is the development and ultimate application of biological substitutes to restore, maintain or improve tissue functions. [Skalak, R. and Fox, C. F., “Tissue Engineering”, Alan R. Liss Inc. N.Y. (1988)]
Collagen is the principal structural protein in the body and constitutes approximately one-third of the total body protein. It comprises most of the organic matter of the skin, tendons, bones and teeth and occurs as fibrous inclusions in most other body structures. Some of the properties of collagen are its high tensile strength; its ion exchanging ability, due in part to the binding of electrolytes, metabolites and drugs; its low antigenicity, due to masking of potential antigenic determinants by the helical structure, and its low extensibility, semipermeability, and solubility. Furthermore collagen is a natural substance for cell adhesion. These properties and others make collagen a suitable material for tissue engineering and manufacture of implantable biological substitutes and bioremodelable prostheses.
As collagen is one major component of these biological substitutes, a method for obtaining sufficient quantities of collagen that is consistent in quality is needed. A need currently exists for an improved method for the removal of non-collagenous components such as cells, cellular debris, and other extracellular matrix components, such as proteoglycans and glycosaminoglycans, normally found in native tissues to yield a substantially pure native collagen matrix. Some of these non-collagenous structures that are present in native tissues are believed to be antigenic and will elicit a chronic inflammatory response when implanted in a host. However, in the art there are a variety of methods for the cleaning of such collagenous tissue which have resulted in collagenous compositions with different characteristics. The method used should be one that maintains the biological and physical properties of collagen and collagenous tissues suitable for use in tissue engineering.
In the art of treating a collagenous tissue to yield essentially a collagenous matrix, detergents and surfactants have customarily been used in the extraction of cells and lipids from the tissue. Detergents such as sodium dodecyl sulfate (SDS) are amphipathic molecules wherein the hydrophobic region binds to protein and are believed to increase the negative charge of the protein. When implanted, the increase in charge results in both the swelling of the tissue due to increased water binding by the hydrophilic region of the molecule, and decreased thermal stability in collagen by disrupting hydrogen bonding. Swelling both opens the structure of the collagen molecule making it susceptible to cellular enzymes such as collagenase and destabilizes the collagen matrix to result in a weakened construct. (Courtman, et al. Journal of Biomedical Materials Research 1994; 28:655-666.) It is further believed that SDS residues remain bound to the collagen and prevent cells from migrating into the implant. (Wilson, G J et al. Ann Thorac Surg 1995; 60:S353-8. Bodnar E, et al. “Damage of aortic valve tissue caused by the surfactant sodium dodecyl sulfate.” Thorac Cardiovasc Surg 1986; 34:82-85.) Because detergents used in a chemical cleaning method can undesirably bind to and alter the bioremodeling capabilities of collagen in the treated tissue, the inventors have developed a method that eliminates the need for detergents.
Chemical cleaning of tissue with enzymes such as trypsin, pepsin and collagenase is known in the art but their use will result in chemical modification of the native collagen molecules and will adversely affect the structural integrity of the construct. Enzyme treatment of collagenous tissue is known in the art for removal and/or modification of extracellular matrix associated proteins. Proteases such as pepsin, trypsin, dispase, or thermolysin are used in the removal of collagen telopeptides to yield atelopeptide collagen. Collagen telopeptides are the non-triple helical portion of the collagen molecule and have been thought by some researchers to be weakly antigenic while by others they are thought to be responsible for the strong mechanical properties of collagen. Limited digestion of collagenous tissue will remove telopeptides without dissociation of the collagen matrix of the tissue, while prolonged digestion will dissociate the collagen fibrils into atelopeptide collagen monomers. It is also known in the art to modify and remove nucleic acids from the matrix using enzymes that digest endogenous RNA and DNA through use of RNAse and DNAse, respectively. As treatment with enzymes can affect the structural integrity of the collagen, the present method of the invention circumvents their use.
Methods for obtaining collagenous tissue and tissue structures from explanted mammalian tissue, and processes for constructing prostheses from the tissue, have been widely investigated for surgical repair or for tissue and organ replacement. The tissue is typically treated to remove potentially cytotoxic cellular and noncollagenous components to leave a natural tissue matrix. Further processing, such as crosslinking, disinfecting or forming into shapes have also been investigated. Previous methods for treating collagenous tissue to remove tissue components from the organized tissue matrix have employed detergents, enzymes or promote uncontrolled swelling of the matrix. WO 95/28183 to Jaffe, et al. discloses methods to decrease or prevent bioprosthetic heart valve mineralization postimplantation. The disclosed methods provide biological material made acellular by controlled autolysis. Autolysis is controllably performed using at least one buffer solution at a preselected pH to allow autolytic enzymes present in the tissue to degrade cellular structural components. U.S. Pat. No. 5,007,934 to Stone and, similarly, U.S. Pat. No. 5,263,984 to Li, et al. both disclose a multiple step method for chemical cleaning of ligamentous tissue. The method utilizes a detergent to remove lipids associated with cell membranes or collagenous tissue. U.S. Pat. No. 5,523,291 to Janzen, et al. discloses an comminuted injectable implant composition for soft tissue augmentation derived from ligamentum nuchae. The ligament is treated with a series soaks in a strongly alkaline solution of sodium hydroxide followed by hydrochloric acid solution and then sodium bicarbonate. U.S. Pat. No. 5,028,695 to Eckmayer, et al. discloses a process for the manufacture of collagen membranes in which collagenous tissue is repeatedly treated with a strong alkali and subsequently with a strong acid for a number of times then further treated with inorganic saline treatment to shrink the membranes and then with solvent to dry them.